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Paper and Oral Presentation Guidelines

The topics covered in the Research Reports are an essential part of the course curriculum, and cover important topics in protein folding, misfolding, aggregation, chaperones and their physiological roles.

Oral Presentation

The goal of the oral presentation is to make the class aware of the subject and problem under study. The oral presentations will be 20 minutes each with each team member giving 10 minutes. A five-minute question and discussion period will follow. You will need to have the abstracts available for the class on the day you are presenting. The final papers are not due until the last week of classes.

Assume you are at a scientific meeting; use Powerpoints or overheads to illustrate key points where convenient. Try to follow the format of: first, pose the question; second, describe the experiments designed to answer the question; third, report the result; and, finally, analyze and interpret the results.

A 10-15 page paper requires 30-45 minutes for oral presentation. Therefore, the live class presentation, though following your manuscript content, needs to be specifically constructed and tuned as an oral presentation. In particular, you need to pull out a key question, critical experiment, its protocol, results, and interpretation.

As in a professional presentation, the presentations should, if possible, be rehearsed and timed beforehand to another member of the class.

Timing

In order to present to the class, much of your reading, assimilation, and integration needs to be done before the day of presentation. However, the actual final written papers are due on the Wednesday of the last week of classes, Ses #38. (Except for the teams presenting on the Ses #38, whose papers will be due 2 days after Ses #38. Student presentations will start on Monday, Ses #30 and continue through Ses #38.

Paper Guidelines

The format of the final papers should be a Mini review of the selected topic. At the point you have completed the paper, you should be an expert on the subject, with the additional good feeling of having made your understanding available to your classmates. The listed references are starting points. You are responsible for finding the latest relevant results, using the tools built in to modern journal on-line resources. Just as with most scientific publications, the paper should be a single coherent integrated text, not the joining together of the writing of each author.

The text should begin with an introduction which gives the background and general context of the work, including the societal and biomedical importance. This is to be followed by a Critical Results and Discussion section which reviews the findings that you consider most relevant and important. Try to follow the format of first posing the question; then describing the experiment designed to answer the question; finally analyzing the resulting data. Reproduce a few figures to illustrate aspects of the subject.

The paper should consist of a title page with an Abstract (<250 words) of the most important results and conclusions. At the end of the Abstract, give the reference to a review article, a very recent research paper, and a paper you consider to be seminal. The abstracts will be copied and distributed to the class.

The length of the text proper should be 10-15 double spaced pages, with 15 pages the absolute limit. The references and figures are additional.

The references should include the full titles of the papers.

The target audience is the other students in the class.

Student Presentation Topics

The references listed are to get you started. They do not represent a complete or systematic sampling of the literature. Finding, reading and assimilating a subset of critical papers is your responsibility.

Class projects.

PROJECT #

TOPICS

DESCRIPTION & CITATIONS

Protein folding in vitro / protein structure and sequences

1

The conformations of aromatic side chains in globular proteins

Most aromatic rings are found in the interior of proteins. What kinds of interactions are aromatic rings involved in within protein interiors? There is evidence in some proteins that the rings can flip. How rapidly? How general is this motion?

Short tripeptides with collagen-like sequences have recently been crystallized and their structure solved by X-ray diffraction. Using 2-D NMR it has been possible to follow the actual kinetics of the chain folding and association reaction and the effects of certain glycine substitutions.

Coiled coils are not limited to pairs; depending on the details of the residues at the buried hydrophobic positions, the helices will form trimeric and higher associations. Recent studies reveal the fine points of the relationship between sequence and tertieary associations.

Electrostatic interations in the folding, association and registration of the coiled coils

Though the hydrophobic interactions are critical, charged side chains interactions also control the structure and association of coiled/coils in both tropomyosin, and leucine zippers. Recent papers report differing values for the contributions of these ion pairs to stability.

How important is the continuity and connectedness of amino acid sequences? In BPTI, the Greek key beta barrel proteins and some larger enzymes, the N- and C- termini have been joined together and new physical ends created, to examine the effects of sequence permutation on chain folding.

Recent studies have begun to identify intermediates in the formation of small beta sheet proteins such as the interleukins and fatty acid binding proteins. These experiments utilize both hygrogen/deuterium exchange combined with spectroscopic procedures.

In many oligomeric proteins assembly proceeds not from native subunits but from folding intermediates of the subunits. One of the best understood cases is the heterdimeric luciferase from the bacteria that illuminate the eyes of deep sea fish.

One of the very few membrane proteins whose three dimensional structure has been solved is the rhodopsin of the visual system. The best defined experiments on how these transmembrane helices associate within the membrane have been done with the bacterial and mammalin opsin.

Functions of prolyl hydroxylases in collagen chain folding and maturation

Prolyl hydroxylase is responsible for the formation of hydroxyproline on newly synthesized chains and is thought to be involved in regulating triple helix formation. Underhydroxylation of prolines is the molecular defect in scurvy, vitamin C deficiency.

What is the role of proline isomerization and proline isomerase in the folding of newly synthesized polypeptide chains within cells, including procollagen? Prolyl isomerase, originally called cyclophilin is the target of the cyclosporin class of immunosuppressive drugs. It turns out to function in many unexpected cellular processes.

Genetic studies in bacteria have identified a family of proteins involved in reduction and oxidation of thiols, and presumed to be involved in the folding of some proteins.

16

The role of protein disulfide isomerase in the endoplasmic reticuluum

How are disulfide bonds formed in newly synthesized polypeptide chains destined for export from eukaryotic cells?

17

In Vivo folding and assembly of the influenza hemagglutinin

The intracellular assembly and maturation of this trimeric viral coat protein is one of the better model systems in eukaryotic cells.

18

The secB chaperonins in proteins destined for export

A number of proteins destined for export (and perhaps folding) outside the cell must be maintained in a non-folded state after synthesis. Some of those are maintained in this state by the secB protein of E.coli, whose mechanism has been studied in considerable detail.

19

Function of the lens chaperone alpha-crystallin

This is a member of the small heat shock chapeorone family. It is present at high coneentratons in the lens and is thought to protect lens crystallins from radiative or oxidative damage leading to cataract.

How does the chaperone actually function in ensuring productive protein folding? How does the Jack in the Box work, how is ATP hydrolysis coupled to chaperone function? What is the relationship of GroES binding and release to folding within the GroEL lumen?

21

Chaperonin function in bacterial pilus assembly

Bacteria use extra cellular flagelli and pili for swimming, attaching to other cells and transporting DNA. The folding and assembly of the proteins for these organelles utilize specialized chaperonins some of which function in the bacterial periplasm. These function both in chain folding and in polymerization of the extracellular pilus organelle.

Many newly synthesized proteins have to transit a membrane, for example, for import into mitochondria or for entry to the endoplasmic reticulum. In general, the polypeptide chains have to be maintained in an unfolded state. The proteins forming these channels have recently been identified in a number of organisms.

23

Function of the DNA K (HSP70) class of chaperonins

These appear to interact with newly synthesized chains at an earlier stage in folding than the GroE class, and function as a complex of DNA K, DNA J, and Grp E. They do not have a lumen, but appear to bind an unfolded peptide in an elongated cleft.

Increased susceptibility to lung damage from smoking and dusts is associated with certain alleles of the anti-elastase that functions in the lung. Recent evidence reveals that their major familial form is due to a defect in the folding of the protein.

Cystic fibrosis is due to a defect in the chloride transporter protein in the respiratory tract. Recent evidence indicates that the most common inherited form is due to a protein folding defect. This is the most developed model of the role of protein folding defects in human disease.

27

Lens crystallins and cataracts

The proteins of the lens, crystallins, have a variety of unusual properties, at least at high concentrations. The three-dimensional fold of the Gamma crystallin has been solved and the protein can be refolded in vitro. Rare inherited cases of juvenile-onset cataract are due to crystals formed in situ. A variety of indirect evidence suggests that mature-onset cataracts represent the aggregation or polymerization of partially folded forms of these proteins.

Topic C: Characterization of the yeasts prion proteins. Sophisticated genetic analysis available in yeast has allowed a very penetrating analysis of the origin and pheotypes of the sup35 yeast prion protein.

30

Light chain amyloidosis

Cancer patients with a form of leukemia called multiple myeloma often accumulate amyloid deposits composed of the overproduced light chains. Aspects of this aggregation reaction have been elucidated through in vitro experiments.

31

Superoxide Dismutase defect in ALS (Lou Gehrig's Disease)

Recent evidence indicates that amylotrophic lateral sclerosis is associated with a defect in the function of the widely distributed protein Superoxide Dismutase. The associated amino acid substitution may affect folding or stability rather than metabolic function.

A rare but well studied class of amyloid diseases are due to deposition of the carrier protein trans-thyretin which is a retinol carrier protein. Features of the in vitro unfolding/ refolding reaction correlate with the conditions that yield pathology.

Loss of function of a number of cellular proteins, which control DNA replication and cell division, is associated with tumor formation. Particularly well-studied are p%3 and p21. There is considerable evidence for the p16 ankyrin proteins that some of these mutations may represent protein folding defects.

34

Protein aggregation in Huntington's disease

The modified protein product associated with CCC expansions in Huntington's disease has recently been identified. The presence of polyglutamine insertions and expansions appears to be a more general source of cellular pathology. An aggregated form of Huntington's is found within the cell nucleus.